Symbiotic bacteria, immune-like sentinel cells, and the response to pathogens in a social amoeba

Some endosymbionts living within a host must modulate their hosts' immune systems in order to infect and persist. We studied the effect of a bacterial endosymbiont on a facultatively multicellular social amoeba host. Aggregates of the amoeba Dictyostelium discoideum contain a subpopulation of sentinel cells that function akin to the immune systems of more conventional multicellular organisms. Sentinel cells sequester and discard toxins from D. discoideum aggregates and may play a central role in defence against pathogens. We measured the number and functionality of sentinel cells in aggregates of D. discoideum infected by bacterial endosymbionts in the genus Paraburkholderia. Infected D. discoideum produced fewer and less functional sentinel cells, suggesting that Paraburkholderia may interfere with its host's immune system. Despite impaired sentinel cells, however, infected D. discoideum were less sensitive to ethidium bromide toxicity, suggesting that Paraburkholderia may also have a protective effect on its host. By contrast, D. discoideum infected by Paraburkholderia did not show differences in their sensitivity to two non-symbiotic pathogens. Our results expand previous work on yet another aspect of the complicated relationship between D. discoideum and Paraburkholderia, which has considerable potential as a model for the study of symbiosis.

resistance to ethidium bromide, despite interfering with the sentinel cells that would otherwise remove such toxins from D. discoideum aggregates. We hypothesized that it might provide a similar protective effect against intracellular pathogens that might otherwise threaten it and its hosts' fitness. In this study, we take advantage of recent advances in our understanding of the diversity of Paraburkholderia to further explore the consequences of its relationship with its D. discoideum hosts.

Culture conditions for Dictyostelium discoideum clones and bacteria symbionts
We used D. discoideum clones collected from Virginia, Texas and North Carolina. We grew D. discoideum clones from frozen spore stocks on SM/5 nutrient agar plates (2 g glucose, 2 g Oxoid bactopeptone, 2 g Oxoid yeast extract, 0.2 g MgSO 4 , 1.9 g KH 2 PO 4 , 1 g K 2 HPO 4 and 15.5 g agar per litre double-distilled water (DDH 2 O)) and food bacteria Klebsiella pneumoniae at room temperature (22°C). We obtained K. pneumoniae from the Dicty Stock Center. Klebsiella pneumoniae was streaked onto SM/5 plates from frozen stocks and allowed to grow until stationary phase. We prepared K. pneumoniae bacterial suspensions with an optical density (OD) of A600 1.50 in KK2 buffer (2.25 g KH 2 PO 4 and 0.67 g K 2 HPO 4 per litre DDH 2 O) using a BioPhotometer (Eppendorf, New York). The D. discoideum clones and specific symbionts used in these experiments are included in table 1. We removed (cured) Paraburkholderia from the infected clones using either ampicillin-streptomycin or tetracycline antibiotic treatment. We verified Paraburkholderia removal using polymerase chain reaction (PCR) with Paraburkholderia specific primers [30].

Visualizing sentinel cells in slug trail assay
To determine if D. discoideum sentinel cell numbers are reduced by Paraburkholderia presence, we used four clones colonized with P. agricolaris and four clones colonized with P. hayleyella. These clones include QS70, QS159, QS161 and NC21 for P. agricolaris and QS11, QS21, QS22 and QS23 for P. hayleyella. We used the same eight clones cured of their Paraburkholderia infections as our uninfected control to compare against the infected D. discoideum clones. We adapted methods from Brock et al. [36] to visualize and collect sentinel cells by staining D. discoideum isolates with a low dose of ethidium bromide (EtBr), an intercalating agent that interferes with nucleic acid synthesis and is commonly used as a fluorescent tag [37]. Using a low dose of ethidium bromide allowed us to visualize sentinel cells, which pick up the chemical and fluoresce, without increasing cell death [36]. However, sentinel cell identify is thus confounded with ethidium bromide treatment (though at a dose that should not have major effects). To prepare ethidium bromide-treated plates, we used 50 × 15 mm Petri plates with non-nutrient agar (9.9 g KH 2 PO 4 monobasic, 1.78 g Na 2 HPO 4 dibasic and 15.5 g agar per litre DDH 2 O) containing 1.0 µg per ml EtBr. Sixty millilitres of non-nutrient agar was poured first and allowed to set. We laid three microscope slides (3 00 × 1 00 × 1 mm) touching each other on top of the cooled agar. Then the slides were embedded in the agar by adding 25 ml of the same non-nutrient agar on top of the slides.
To set up the migration plates, we prepared a concentrated K. pneumoniae bacteria suspension. We used an overnight bacterial culture started from a single colony, and grown in Luria broth (10 g tryptone, 5 g Oxoid yeast extract and 10 g NaCl per litre DDH 2 O) shaking at 25°C. Next day, we pelleted the overnight culture by centrifugation at 10 000g for 5 min at 4°C discarding the supernatant. The bacterial pellet was resuspended and washed in KK2 buffer (2.25 g KH 2 PO 4 and 0.67 g K 2 HPO 4 per litre DDH 2 O). We resuspended the final pellet in a small volume of KK2. The bacterial suspension was diluted accordingly to obtain an OD A600 of 35.00 using a BioPhotometer (Eppendorf, New York).
We collected D. discoideum spores in KK2 buffer and determined the spore count using a haemacytometer and light microscope. We suspended 2.0 × 10 5 spores in 200 µl of prepared bacterial suspension, and 50 µl of the mixture was dispensed in a line parallel to the embedded microscope slides on one edge of the plate. The spore mixture was allowed to dry. The plate was wrapped in aluminium foil with a small hole poked on the opposite side of the line where the spores were deposited. The plates were placed so the hole faced a source of light, towards which the slugs would migrate, and were stored at room temperature (22°C) for 168 h to allow for sufficient slug migration across the ethidium bromide starving agar Petri plate.
To visualize sentinel cells present in slug trails, we excised the embedded microscope slides and placed microscope slide coverslips (24 × 60 mm) on top of the slug trails on the agar. These trails were imaged using a Nikon A1Si laser scanning confocal microscope (Nikon, Tokyo), at 10× magnification under UV light (Texas red filter, λ = 561.3 nm). We used the 'Scan Large Image' function in the software program (NIS Elements Advanced Research v. 4.12.01) to capture an image of the slug trails over a large span of area. This function captures multiple images over the selected area and stitches the images together (10% blend). We then used the 'Annotations and Measurements' tool to measure the length of the sectioned trails from which sentinel cells were counted. Present in the slug trails were both single and clumped groups of sentinel cells. We counted sentinel cells in clumped groups as an estimate based on the size of one sentinel cell.

Colonizing uninfected hosts with Paraburkholderia and migration assay
To test if colonization with Paraburkholderia affects sentinel cell number, we first needed to determine what proportion of Paraburkholderia to mix with the food bacteria K. pneumoniae. Previously, we had determined that sufficiently high infectious doses of Paraburkholderia are toxic enough to prevent hosts from forming slugs or fruiting bodies (data not shown).
We prepared bacterial suspensions of K. pneumoniae, P. agricolaris BaQS159 and P. hayleyella BhQS11 at OD600 = 35.00 using the method described above. We then combined suspensions of food bacteria and either P. agricolaris or P. hayleyella at different ratios to compare the effects of different infectious doses. We performed 10-fold serial dilutions to test for slug formation from 1% Paraburkholderia + 99% K. pneumoniae down to 0.0001% Burkholderia + 99.9999% K. pneumoniae. The low percentage of Paraburkholderia was achieved by diluting the Paraburkholderia bacterial suspension down to a lower OD such that sufficient volume could be pipetted. We did not obtain adequate slug formation until we reduced the percentage of Paraburkholderia to 0.001% and 0.0001%. We followed the same steps to plate spores on ethidium bromide plates as described in the visualizing sentinel cells assay above. We used 100% K. pneumoniae as our control.
After the slugs were allowed to migrate and fruit, we tested sori to determine if bacteria carriage was induced successfully in naturally uninfected clones. We adapted methods of the spot test described in royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230727 Brock et al. [34]. From the fruiting bodies formed at the ends of the slug trails, we randomly picked up individual sori using a filtered pipette tip. Each sorus was transferred onto SM/5 nutrient agar plates as individual spots. We incubated the plates at room temperature (22°C) for 2 days, examined for bacteria growth, and recorded the number of positive spots of bacterial growth.

Bead uptake assay
To determine if Paraburkholderia-infected sentinel cells are able to function as well as those from uninfected hosts, we counted the uptake of 0.5 µm diameter fluorescent latex beads in sentinel cells present in disassociated slugs. We used three types of host clones consisting of four naturally uninfected, three infected by P. agricolaris, and three infected by P. hayleyella. To remove extracellular bacteria, we washed log phase amoebae with KK2 buffer. These amoebae were then allowed to develop on filters until they formed aggregates. We collected aggregates with a pipette tip and disassociated cells by pipetting the collected aggregates several times. Disassociated cells were then mixed with fluorescent beads at a 1 : 10 ratio of cells to beads. Sentinel cells were identified by their ability to take up beads. We counted the number of beads present in each of 10 sentinel cells selected haphazardly for each D. discoideum clone in each set for a total of 100 sentinel cells.

Pathogen fitness assay
To assess fitness effects associated with carriage of Paraburkholderia, we chose two pathogenic bacteria species known to infect D. discoideum intracellularly (Staphylococcus aureus Rosenbach (Wichita) ATCC 29213, and Salmonella enterica ATCC 14028) [38,39]. For this assay, we tested four hosts naturally infected with P. agricolaris, four hosts cured of their P. agricolaris, four hosts naturally infected with P. hayleyella, four hosts cured of their P. hayleyella, and four naturally uninfected hosts. Table 1 for specific clone identities. Each clone was grown on either 100% St. aureus (Gram-positive pathogen), Sa. enterica (Gram-negative pathogen), or K. pneumoniae (good-food control). We used total spore production as our measure of host fitness. To set up each assay, we plated 2 × 10 5 spores of each clone in each condition onto SM/5 agar plates in triplicate. All clones formed fruiting bodies within 2-3 days. We collected spores separately from two of the plates 5 days after fruiting using the method previously described in Brock et al. [34]. Briefly, we collected spores by washing plates with KK2 buffer supplemented with 0.01% NP-40 alternative (Calbiochem). Then, we counted spores using a haemacytometer and a light microscope.

Statistical analyses
We performed statistical analyses in R (v. 3.6.3). To compare the means of different groups, we fit models followed by pairwise contrasts calculated with the emmeans package [40] using fdr to adjust for multiple comparisons. Sentinel cell data was collected from slug trails with multiple measures from each trail and multiple trails for each clone. To account for this nested structure of sentinel cell counts, we included the random effects of trail nested within clone in linear mixed models (LMM) using the nlme package [41]. We also log transformed sentinel cell counts to reduce the skew from high counts. For our bead results, we fit generalized linear mixed models (GLMM) in the lme4 package [42] with a Poisson link function and clone as a random effect.
To measure how pathogens affected spore production, we used a generalized least-squares (GLS) model. Because infections with different pathogens resulted in groups with different variances, we weighted observations using the varIdent function in the nlme package. To reanalyse the spore data in Brock et al. [36] that tested the effect of a high dose of ethidium bromide, we used a LMM. To account for the two technical replicates used in this experiment, we included clone as a random effect. We scaled spore production values by subtracting the mean and dividing by the standard deviation.
For both our pathogen and ethidium bromide models, we estimated effects relative to the control condition (grown with K. pneumoniae food bacteria or without ethidium bromide), where hosts are not expected to suffer reduced spore production. This effect relative to the control will also capture the inedibility of pathogens, which is a consequence of pathogens being able to evade phagocytosis [43]. We are thus measuring the effect of some stressor (infection/inedibility or toxin) relative to healthy hosts. We are mostly interested in the interaction effects between Paraburkholderia infection status (infected or cured) and stressors ( pathogen infection or toxins). These effects would indicate that Paraburkholderia infection (or curing) protects or causes increased harm for hosts when exposed to a stressor.

Is the function of sentinel cells from infected hosts impaired?
Because hosts infected with Paraburkholderia have fewer sentinel cells (figure 1), we suspected Paraburkholderia infection may also impact sentinel cell function. To test sentinel cell function, we measured the number of beads that sentinel cells were able to phagocytose. We found that sentinel cells from P. agricolaris-infected hosts take up fewer beads (about 17% less) compared with uninfected

How does Paraburkholderia infection affect host response to toxicity and pathogens?
A prior study found that Paraburkholderia infection protected hosts when they were exposed to a toxically high (more than 10 times the dose used in this study) amount of ethidium bromide [36]. However, hosts in their natural soil environment are unlikely to come in contact with ethidium bromide. We instead sought a more natural stressor to understand how reduced sentinel cell function affected hosts. We suspected that reduced function of sentinel cells due to Paraburkholderia infection may make infected hosts more susceptible to harm from other pathogens. We first reanalysed the data from Brock et al. [36] where infected, uninfected and cured hosts were grown on starving agar plates with or without toxic ethidium bromide (figure 3a). While the original study included hosts infected by both P. agricolaris and P. hayleyella, it did not draw a distinction between the two species. (Additionally, the dataset only included cured controls for strains infected by P. hayleyella). We estimated the effects on host spore production of ethidium bromide, infection, curing, and the interactions between these categories relative to uninfected controls that were not exposed to ethidium bromide. We describe the estimated effects from top to bottom in figure 3b. We found that ethidium bromide (EtBr) lowered host spore production (  , d.f. = 59) produced more spores when exposed to ethidium bromide than expected from the separate effects of Paraburkholderia infection and ethidium bromide. This effect is due to Paraburkholderia infection, at least for P. hayleyella, as these hosts cured of their symbionts did not deviate from the additive effects of curing and ethidium bromide (Ph cured Ã EtBr, estimate = −0.681, 95% CI = [−1.523, 0.160], d.f. = 59).
To address the possibility that Paraburkholderia infection makes hosts more prone to harm from a more natural stressor, we tested spore production in the presence of two pathogenic bacteria, Salmonella enterica and Staphylococcus aureus. We used Klebsiella pneumoniae as our control food bacteria for comparison. To measure the harm of infection with these other pathogens, we counted the spores produced by D. discoideum (figure 4a). We asked two questions: (i) Does infection with pathogens lower D. discoideum spore production relative to growing on food bacteria? (ii) Does infection with Paraburkholderia affect harm to hosts when infected with other pathogens?
To answer these questions, we fit a generalized least-squares model with interaction terms between each pathogen infection status (either Sa. enterica or St. aureus) and each Paraburkholderia infection status (either P. agricolaris, cured of P. agricolaris, P. hayleyella or cured of P. hayleyella) to D. discoideum spore production measures (figure 4a). We used this model to estimate the effects of pathogen infection, Paraburkholderia status, and co-infections (interactions) relative to uninfected D. discoideum that were grown on only food bacteria (figure 4b). Working down from the top of figure 4b,   Co-infection by Paraburkholderia and a pathogen did not increase or decrease harm from pathogens beyond the additive effects of both kinds of infection (interaction terms with Ã s overlap 0). These results show that Paraburkholderia and pathogen infections both reduce D. discoideum spore production, but that Paraburkholderia does not make hosts more susceptible to harm by pathogens. On the other hand, Paraburkholderia also offers no protection against the pathogens we tested in contrast to the results with ethidium bromide.

Discussion
In this study, we investigated the effects of Paraburkholderia infection on the sentinel cells of D. discoideum hosts. Due to sentinel cells' presumed function as a primitive immune system within D. discoideum aggregates, we also explored the consequences of Paraburkholderia infection on D. discoideum's sensitivity to toxins and pathogens.
Dictyostelium discoideum aggregates produce a subpopulation of sentinel cells which seem to serve as an innate immune system for the multicellular stages of its life cycle [24]. Like the cellular immune systems of more familiar multicellular organisms, sentinel cells circulate within D. discoideum aggregates, sequestering and disposing of potentially hazardous foreign material like toxins or pathogens. In a previous study, we observed that some wild D. discoideum isolates carrying certain Paraburkholderia bacteria through their social cycles also produced significantly fewer sentinel cells [36].  royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230727 To explore the role of D. discoideum's intracellular symbiont Paraburkholderia on its host's sentinel cells, we compared the number of sentinel cells produced in slugs made by wild D. discoideum strains known to harbour P. agricolaris or P. hayleyella symbionts with slugs made by the same strains after antibiotic treatment. We found that antibiotically treated D. discoideum, cured of their normal symbionts, produced significantly more sentinel cells than infected cells (figure 1). When we then reinfected these cured strains with P. agricolaris or P. hayleyella in the laboratory, the effect was reversed and fewer sentinel cells were produced. These results establish a causal link between infection by Paraburkholderia and the previously observed reduced sentinel cell numbers in infected D. discoideum strains. Paraburkholderia infections reduced D. discoideum sentinel cell production even at very small infectious doses.
In addition to quantifying the reduction in sentinel cell numbers in infected D. discoideum, we also tested sentinel cell functionality by measuring sentinel cells' ability to sequester fluorescently labelled beads. We found that individual sentinel cells produced by infected D. discoideum sequestered significantly fewer beads than sentinel cells from uninfected D. discoideum (figure 2). We suspect that D. discoideum sentinel cells take up fewer beads because Paraburkholderia infection reduces their function. However, we cannot completely rule out that external Paraburkholderia are affecting bead engulfment. During the disassociation step, where D. discoideum cells in aggregates are separated, Paraburkholderia could be released. If these bacteria bind to beads and affect phagocytosis this could also explain our results. However, we think this explanation is less likely than infection lowering sentinel cell function. External bacteria should be rare in our experiments because infection doses were low and experiments were performed on filters without nutrients for bacterial proliferation.
Our results suggest that D. discoideum infected by Paraburkholderia not only produces fewer sentinel cells, but those that it does produce are less functional. Insofar as sentinel cells serve an important role in clearing potentially damaging foreign substances from multicellular D. discoideum aggregates prior to the production of fruiting bodies, we expected infected D. discoideum to have impaired immune function and be more sensitive to toxins or pathogens. To test this, we first reanalysed data measuring the toxic effect of high-dose ethidium bromide on spore production in infected and uninfected D. discoideum [36]. Uninfected D. discoideum exposed to toxic amounts of ethidium bromide produces fewer spores during its fruiting stage (figure 3), presumably reflecting death or reduced functionality of cells within the aggregate. By contrast, however, D. discoideum infected by Paraburkholderia produced similar numbers of spores with and without the presence of ethidium bromide. While infected D. discoideum produce fewer spores overall due to the effects of Paraburkholderia infection itself [30], they no longer appear to be sensitive to ethidium bromide's toxic effects. One potential explanation for this reduced toxicity is that Paraburkholderia themselves are taking up ethidium bromide and reducing exposure to their hosts.
Sentinel cells may represent an adaptation against threats posed by pathogens-either through infection or via the production of toxins. Dictyostelium discoideum is known to be susceptible to a wide variety of pathogens [44], which, combined with its similarities to human macrophages, has made it a common model for the study of pathogenesis and immunity [45]. Previous work suggests that sentinel cells probably serve a protective function against pathogens, presumably by sequestering and removing them from D. discoideum slugs as they do with ethidium bromide [24]. Sentinel cells were shown to upregulate their expression of the tirA and tirB genes, apparent homologues of genes known to be involved in animal and plant innate immunity signalling. Mutant strains lacking functional TirA showed increased sensitivity to the virulent pathogen Legionella pneumophila.
In the light of sentinel cells' potential immune function, we explored whether Paraburkholderia's effects on sentinel cell number and functionality increased D. discoideum's sensitivity to other pathogens, as would be expected if sentinel cell activity played a key role in immunity. We exposed uninfected amoebae and amoebae carrying Paraburkholderia symbionts to Staphylococcus aureus and Salmonella enterica, two pathogens known to infect D. discoideum [38,39]. Unsurprisingly, D. discoideum grown on either pathogen produced fewer spores relative to more edible bacteria, reflecting St. aureus and Sa. enterica's negative effects. Despite its effects on sentinel cell number and functionality, however, infection by Paraburkholderia did not have a significant effect on D. discoideum's sensitivity to either tested pathogen (figure 4).
Our results suggest that D. discoideum infected by Paraburkholderia experiences reduced sentinel cell production and function. Building upon previous work, this study demonstrates that Paraburkholderia itself is responsible for changes in sentinel cells (rather than some strains of D. discoideum evolving reduced sentinel cells to facilitate symbiosis or for some other purpose). Naive D. discoideum strains with no known association with Paraburkholderia produce fewer sentinel cells when infected, and infected D. discoideum sentinel cell production is restored when Paraburkholderia symbionts are cleared by antibiotic treatment.
It is unclear whether Paraburkholderia's effect on its host's sentinel cells is adaptive. Given that some evidence suggests sentinel cells may be involved in clearing bacteria from D. discoideum slugs, it is intuitive to speculate that Paraburkholderia could benefit from inhibiting them. Alternatively, Paraburkholderia's effects on sentinel cells may be pleiotropic consequences of other traits. Paraburkholderia's intracellular lifestyle is probably only possible because it can waylay the mechanisms by which D. discoideum phagocytoses and destroys its prey. A recent study [46] has shown that Paraburkholderia inhibit D. discoideum phagosomes from becoming acidic, potentially preventing digestion. If phagocytosis is also key to sentinel cell function during the slug stage, whatever mechanism Paraburkholderia uses to survive phagocytosis during its host's vegetative stage may inhibit sentinel cell function as a side effect.
Despite its effect on sentinel cell number and function, Paraburkholderia infection also has the apparently paradoxical effect of reducing its host's sensitivity to the toxin ethidium bromide. One possible explanation is that Paraburkholderia may neutralize ethidium bromide in a way which compensates for the presumed loss of D. discoideum's own defences resulting from fewer, less functional sentinel cells. Many bacteria have metabolic capabilities unavailable to eukaryotes, and these form the basis for various other symbioses [47][48][49]. Previous work has suggested that Paraburkholderia and D. discoideum have a complex relationship with both antagonistic and cooperative elements, and it is likely that whether Paraburkholderia is a beneficial partner or a parasite depends on the specific context. If Paraburkholderia can protect its host from toxins, then the likelihood of encountering these toxins could play a role in whether Paraburkholderia behaves as a cooperative symbiont or a parasite.
By contrast, we did not observe any effect of Paraburkholderia infection on D. discoideum's sensitivity to the pathogens St. aureus or Sa. enterica. A previous study demonstrated that D. discoideum mutants lacking a functional tirA gene-normally highly expressed in sentinel cells-were extremely sensitive to virulent L. pneumophila [24]. This has been taken to imply an important role for sentinel cells in protecting D. discoideum from pathogens, but our results cast some doubt on this interpretation. Paraburkholderia-driven reductions in sentinel cell number and functionality did not seem to render D. discoideum any less capable of surviving St. aureus or Sa. enterica as would be expected if they served a vital immune function against these pathogens. It may be that sentinel cells' potential immune function is context specific, or that D. discoideum combats different pathogens in different ways. Alternatively, perhaps infected D. discoideum with fewer and less functional sentinel cells are more sensitive to pathogens but are compensated for somehow by the presence of Paraburkholderia. This compensation could result from competition between Paraburkholderia and pathogens that reduces pathogen density. Similar benefits from bacterial interactions inside hosts have been suggested for numerous other systems [50,51].
The specific role of D. discoideum's sentinel cells in defending against specific pathogens is not yet fully clear, but we consider it likely that they function as a simple immune system for the multicellular portions of D. discoideum's life cycle. Previous studies demonstrate that sentinel cells sequester and discard toxic ethidium bromide, which may suggest a more general role in combatting toxins produced by other microbes in D. discoideum's environment. If sentinel cells do have an immune function, then Paraburkholderia's ability to reduce sentinel cell production and functionality may be an adaptation to actively interfere with its host's defences.
Regardless of whether Paraburkholderia acts as a pathogen or a mutualist, this sort of immune interference has precedence in other systems. Pathogens, perpetually locked in antagonistic coevolutionary arms races with their hosts, have evolved myriad ways of interfering with every part of their hosts' immune systems [52,53]. While this sort of antagonism might be expected between foes, it is also known to occur in apparently cooperative interactions. Research on other host/ symbiont systems ranging from plants and their root symbionts [54] to animals and their symbionts [55][56][57][58][59] suggests that modulation of the host's immune system-whether imposed by the host itself or by interference from the symbiont-is often critical for establishment and persistence of symbioses.
Paraburkholderia's effect on D. discoideum's sentinel cells adds an intriguing element to our understanding of their already complex relationship. This study has identified an additional cost of being infected by Paraburkholderia: reduced sentinel cell function. Further exploration into the mechanisms by which Paraburkholderia infects and persists within D. discoideum will help further develop the system as a model for symbiosis, cooperation and antagonism, and provide insights into how immune systems respond to symbionts.
Ethics. This work did not require ethical approval from a human subject or animal welfare committee. Data accessibility. All data and code for this project are included as electronic supplemental material [60] and publicly available at https://gitlab.com/treyjscott/sentinel_cell_project.
Declaration of AI use. We have not used AI-assisted technologies in creating this article.